Composite Milk Samples Research Articles

Prevalence, Risk Factors, and Antimicrobial Resistance of Staphylococcus and Streptococcus Species Isolated from Subclinical Bovine Mastitis.

Subclinical mastitis (SCM) is a prevalent serious disease among dairy cows worldwide. It poses a significant challenge to the dairy industry, animal welfare, and a threat to public health. The present study aimed to investigate the molecular detection, prevalence, and antimicrobial resistance of Staphylococcus spp. and Streptococcus spp. isolated from raw composite milk samples obtained from SCM dairy cattle in Bangladesh. A total of 612 quarters milk samples obtained from 153 cows were analyzed for SCM using the California Mastitis Test. Bacterial isolation and identification were carried out and bacterial species were confirmed using molecular polymerase chain reaction methods. Antimicrobial susceptibility testing was performed using disc diffusion method. The findings revealed that the prevalence of SCM was 70.3% (26/37), 35.95% (55/153), and 23.04% (141/612) in the herd, cow, and quarter levels, respectively. Among the positive samples, 92.7% (51/55) were Staphylococcus spp. (S. aureus, S. chromogenes, and S. simulans) and the remaining isolates were 7.3% (4/55) Streptococcus spp. (Streptococcus agalactiae and Streptococcus dysgalactiae). The most prevalent species was S. chromogenes, accounting for 67.3% (37/55). Antimicrobial susceptibility testing showed that 65.5% of isolates were susceptible to cefoxitin, whereas, 89.1% were resistant to penicillin. Overall, 12 isolates (21.8%) out of 55 were resistant to more than three classes of antimicrobials and were defined as multidrug-resistant isolates. Methicillin-resistance gene was detected in 61.1% of the cefoxitin-resistant isolates. A multivariate logistic regression analysis identified five potential risk factors including the lack of post-milking teat disinfection (OR: 3.06), absence of immediate feeding after milking (OR: 9.81), poor udder hygiene (OR: 7.83), tick infestation (OR: 13.76), and absence of dry cow therapy (OR: 3.31). The findings of the current study underscore the urgent requirement for targeted interventions, considering the identified factors to effectively manage and control SCM in dairy cows.

Relationship between chronic diseases, hair cortisol concentration and welfare of housed dairy goats

The aim of this study was to evaluate the relationship between seroprevalence of chronic diseases, hair cortisol concentration (HCC), and welfare of dairy goats housed throughout a productive cycle. Sixty multiparous dairy goats, over four years old, were selected. An animal welfare assessment was conducted using health indicators for goats, according to the AWIN protocol. Blood samples were also collected for haematology and determination of seroprevalence of chronic diseases, hair samples for determination of HCC, milk samples for chemical composition and somatic cell counts, and faecal samples for parasite load. Small Ruminant Lentivirus (SRLv) had a prevalence of 71.66%, Mycobacterium avium subspecies paratuberculosis (MAP) of 5%, Leptospira interrogans of 40% and Ovine Gammaherpesvirus type 2 (OvHV-2) of 50%. The percentages of goats that tested positive for one, two or three diseases were 31.67%, 50% and 11.66%respectively. Haematological alterations included hyperproteinaemia (84.94 ± 1.58 g/L) and hyperfibrinogenaemia (6.11± 0.65 g/L) for those with one or two diseases, with significant differences being found (P < 0.05). The welfare indicatorsrelated to health and the number of diseases were poor body condition, poor coat, poor udder conformation, andmucosal lesions (P < 0.05). However, no significant differences were observed between HCC and the number of chronicdiseases in dairy goats (P > 0.05). Higher concentrations of cortisol in hair were found at 150 days of lactation (16.65 ± 1.39pg/mg) compared to the mating season (9.55 ± 0.04 pg/mg) (P < 0.05). No associations were found (P > 0.05) betweenthe production, composition, and somatic cell counts in milk and cortisol concentrations and diseases. It was concludedthat the presence of chronic diseases in goats did not influence hair cortisol concentrations, possibly due to an effect ofadaptive tolerance to diseases, as occurs in other domestic species; however, there was an effect of the productive stage.

Effects of Early Lactation Milking Frequency in an Automated Milking System on Cow Performance.

Automated milking systems (AMS) are increasingly adopted for dairy cow production, promoting individualized cow management dependent on factors like lactation stage, age, and productivity. The study objective was to investigate the effects of early lactation milking frequency on cows milked via AMS. Multiparous Holstein cows blocked by parity and due date were randomly assigned to treatments (n = 8 per treatment): three (3X) or six (6X) milkings per day (MPD). The experimental phase (EXP) was defined as 4 to 29 days in milk (DIM). The AMS settings were programed so 3X cows were limited to three MPD while 6X cows were allowed six MPD. Afterwards was the carry over phase (CO) ranging from 30 to 90 DIM; all cows were allowed up to six MPD. Measurements by the AMS included bodyweight, milk yield (MY), and pellet intake. Weekly composite milk samples were analyzed for macronutrient composition and fatty acid (FA) profile. Coccygeal blood was sampled at 3, 8 ± 1, and 13 ± 1 DIM; concentrations of blood plasma analytes were quantified. Greater MPD was achieved for 6X cows versus 3X cows during EXP, but similar during the CO. Daily MY was non-separable during the EXP while 6X cows in their third or greater lactation group (3 + LG) had greater MY than 3X cows of the same LG during the CO. Milk fat content and 4% fat-corrected MY were both greater for 6X, 3 + LG cows during the EXP compared to 3X, 3 + LG cows. Milk FA methyl esters (FAME) proportions were different between MPD groups, with 6X, 3 + LG cows having the lowest short, even-chain FA from de novo or post-absorptive origin. Differences in analytes indicated that 6X, 3 + LG cows experienced metabolic stress and incorporated greater FA from adipose tissue. Greater early lactation MPD in AMS may shift cow nutrient partitioning to support greater production in 3+ parity cows.

A randomized controlled trial to study the effect of supplemental premilking stimulation on milking performance, teat tissue condition, udder health, and well-being in dairy cows

The objectives of this study were to evaluate the effect of supplemental premilking stimulation, provided after manual stimulation, by means of high-frequency pulsation without reduction of the vacuum in the pulsation chamber on milking performance, teat tissue condition, udder health, and well-being in dairy cows. In a randomized controlled trial, Holstein cows (n = 491) from 1 commercial dairy farm with a thrice-per-day milking schedule were assigned to treatment and control groups over a 64-d period. Treatments consisted of a maximum of 20 s of pulsation stimulation at a pulsation rate of 100 (SPS100) or 300 (SPS300) cycles per minute and a pulsation ratio of 25:75, which were applied after completion of manual premilking stimulation upon milking unit attachment. Cows in the control group (CON) received only traditional premilking stimulation by manual forestripping for 6 s, and wiping. Milking characteristics were measured with on-farm milk flow meters. Milking machine-induced short-term (swelling at teat base, firmness at teat end, and teat discoloration) and long-term (teat-end callosity) changes to the teat tissue were assessed manually and visually. Composite milk samples were analyzed for somatic cell counts. Cow hind-leg activity was assessed with 3-dimensional accelerometers. Fecal 11,17-dioxoandrostanes (11,17-DOAs; a group of cortisol metabolites) were determined on wk 4 and 8 to assess the well-being of the cows. Generalized linear mixed models were used to study the effect of treatments on the outcome variables milk yield/milking and milking unit-on time. We observed no meaningful differences among groups for milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the SPS100, SPS300, and CON groups were 13.9 (13.3-14.5), 14.0 (13.4-14.6), and 13.9 (13.3-14.6) kg for milk yield and 218 (212-224), 218 (211-224), and 218 (212-224) s for milking unit-on time, respectively. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.30 (0.95-1.78) for the SPS100 group and 1.50 (1.10-2.05) for the SPS300 group. The odds of long-term changes were 0.94 (0.67-1.34) for cows in the SPS100 group and 0.71 (0.49-1.04) for cows in the SPS300 group. We observed no differences in SCC. In reference to the CON group, the hazard ratio (95% CI) in SPS100 and SPS300, respectively, were 0.35 (0.13-0.98) and 1.22 (0.57-2.64) for clinical mastitis, and 0.34 (0.12-0.95) and 1.28 (0.60-2.73) for culling. The least squares means (95% CI) of hind-leg activity during milking were 8.3 (6.5-10.5), 10.6 (8.1-13.7), and 9.1 (7.2-11.6) movements per milking for the SPS100, SPS300, and CON groups, respectively. The least squares means (95% CI) of fecal 11,17-DOAs concentration (ng/g) at the first and second test days, respectively, were 31.1 (28.1-34.2) and 22.3 (19.2-25.4) for the SPS100 group, 26.4 (23.4-29.4) and 25.2 (22.0-28.4) for the SPS300 group, and 24.8 (21.8-27.9) and 25.0 (21.7-28.3) for the CON group. We conclude that applying supplemental stimulation after manual stimulation through the high-frequency pulsation system tested here did not impart additional benefits to the milk harvesting process, teat tissue condition, somatic cell count, or the well-being of the animals.

Influence of supplemental choline on milk yield, fatty acid profile, and weight changes in postpartum ewes and their offspring.

The most intensive nutritional requirements occur during milk production's peak. Ewe milk contains more protein and fat than cow milk. The nutritional factors significantly determine the composition. The liver undergoes high stress during lactation but is relieved by essential nutrients. Choline acts metabolically as a lipotrope. This compound functions in cell structure construction, maintenance, and acetylcholine synthesis. The animal nutrition industry provides choline from various sources, such as synthetic and natural kinds. This study evaluated the influence of two distinct choline sources on dairy ewes' peripartum and postpartum milk production, composition, and offspring growth. Twenty-four Rambouillet ewes, each weighing around 63.7 ± 1.7 kg, aged three with two previous births, spent 30-day pre-partum and post-partum in individual pens (2 × 2 m). They were given different experimental treatments 30 days before and after birth according to a randomized design; no choline (a), 4 g/day rumen-protected choline (RPC) (b), or 4 g/day thiocholine (c). Milk samples for milk composition and long-chain fatty acid (FA) analysis were taken every 30 days during milk collection. Significant differences (p < 0.05) in ewe body weight, lamb birth weight, and 30-day-old lamb body weight were observed at lambing and on day 30 of lactation due to choline treatment. Milk yield was significantly higher (1.57 kg/day) compared to the control (1.02 kg/day) and RPC (1.39 kg/day), due to the herbal choline source. There was no significant difference in the milk's protein, lactose, fat, non-fat solids, and total milk solids content between the treatments. Herbal choline lowers (p < 0.05) the concentrations of caproic, caprylic, capric, lauric, and myristic acids while boosting (p < 0.05) those of oleic and cis-11-eicosenoic acid, the changes influencing long-chain FA levels (p < 0.05). Providing choline from both sources to ewes enhanced milk production and body weight at lambing and on 30-day post-lambing. The herbal choline supplement altered short-chain milk FAs, while representative concentration pathways affected medium-chain ones.

Validity of luminometry and bacteriological tests for diagnosing intramammary infection at dry-off in dairy cows

The objective of this cross-sectional study was to estimate the validity of laboratory culture, Petrifilm and Tri-Plate on-farm culture systems, and luminometry to correctly identify IMI at dry-off in dairy cows, considering all tests as imperfect. From September 2020 until December 2021, we collected composite milk samples from cows before dry-off and divided them into 4 aliquots for the luminometry test, the Petrifilm (aerobic count), the Tri-Plate, and the laboratory culture. While we assessed multiple thresholds of relative light units (RLU) for the luminometry, we used thresholds of ≥100 cfu/mL for the laboratory culture, ≥ 50 cfu/mL for the Petrifilm, and ≥1 cfu for the Tri-Plate. We fitted Bayesian latent class analysis (LCA) models to estimate the sensitivity (Se) and specificity (Sp) for each test to identify IMI, with 95% credibility interval (BCI). Using different prevalence measures (0.30, 0.50, and 0.70), we calculated the predictive values (PV) and misclassification cost terms (MCT) at different false-negative to false-positive ratios (FN:FP). A total of 333 cows were enrolled in the study from one commercial Holstein herd. The validity of the luminometry was poor for all thresholds, with Se of 0.51 (95% BCI = 0.43-0.59) and Sp of 0.38 (95% BCI = 0.26-0.50) when using a threshold of ≥150 RLU. The laboratory culture had Se of 0.93 (95% BCI = 0.85-0.98) and Sp of 0.69 (95% BCI = 0.49-0.89), the Petrifilm had Se of 0.91 (95% BCI = 0.80-0.98) and Sp of 0.71 (95% BCI = 0.51-0.90), and the Tri-Plate had Se of 0.65 (95% BCI = 0.53-0.82) and Sp of 0.85 (95% BCI = 0.66-0.97). Bacteriological tests had good PVs, with comparable positive PV for all 3 tests, but lower negative PV for the Tri-Plate compared with the laboratory culture and the Petrifilm. For a prevalence of IMI of 0.30, all 3 tests had similar MCT, but for prevalence of 0.50 and 0.70, the Tri-Plate had higher MCT in scenarios where leaving a cow with IMI untreated is considered to have greater detrimental impacts than treating a healthy cow (i.e., FN:FP of 3:1). Our results showed that the bacteriological tests have adequate validity to diagnose IMI at dry-off, but the luminometry does not. We concluded that, while luminometry is not useful to identify IMI at dry-off, the Petrifilm and Tri-Plate tests performed similarly to the laboratory culture, depending on the prevalence and the importance of the FP and FN results.

Prevalence and Antimicrobial Resistance of Escherichia coli O157:H7 and Salmonella, and the Prevalence of Staphylococcus aureus in Dairy Cattle and Camels under Pastoral Production System.

Escherichia coli O157:H7, Salmonella and Staphylococcus aureus are common foodborne pathogens. We determined the prevalence of E. coli O157:H7 and Salmonella in feces and milk and the prevalence of S. aureus in milk from dairy cattle and camels in the Borana pastoral community in the Southern Oromia Region of Ethiopia. Paired individual cow composite (pooled from all quarters in equal proportions) milk and fecal samples were collected from cows (n = 154) and camels (n = 158). Samples were cultured on bacterial isolation and identification media. E. coli O157:H7 and Salmonella isolates were further tested for susceptibility against nine antimicrobial drugs. Different risk factors associated with hygienic milking practices were recorded and analyzed for their influence on the prevalence of these bacteria in milk and feces. The prevalence of E. coli O157:H7 and Salmonella in feces were 3.9% and 8.4%, respectively, in cows, and 0.6% and 2.5%, respectively, in camels. E. coli O157:H7 and Salmonella were detected in the composite milk samples of 2.6% and 3.9% of the cows, respectively, and 0% and 1.3% of the camels, respectively. S. aureus was detected in composite milk samples of 33.4% of the cows and 41.7% of the camels. All E. coli O157:H7 (n = 11) and Salmonella (n = 25) isolates from both animal species and sample types were resistant to at least one antimicrobial drug. Multidrug resistance was observed in 70% (7/10) of the E. coli O157:H7 fecal and milk isolates from cows and 33.3% (2/6) of the Salmonella fecal and milk isolates from camels. The prevalence of these bacteria in feces and milk was not affected by risk factors associated with milking practices. Given the very close contact between herders and their animals and the limited availability of water for hand washing and udder cleaning, these bacteria are most likely present in all niches in the community. Improving community awareness of the need to boil milk before consumption is a realistic public health approach to reducing the risk of these bacteria.

Aflatoxins Contamination in Animal Feeds and Fresh Milk in Kondoa District, Tanzania

Abstract: The occurrence of aflatoxins in animal feeds and milk is common around the World as most livestock keepers are not aware of aflatoxins.&#x0D; Aims: This study was conducted to assess storage and feeding practices, and assessment of aflatoxin B1 and M1 contamination in animal feeds and fresh milk samples in Kondoa.&#x0D; Study Design: The study involved a survey using semi-structured questionnaires and Laboratory analysis of aflatoxin B1 and aflatoxin M1 in animal feeds and fresh milk samples collected from among smallholder dairy farmers.&#x0D; Methodology: Purposive selection of 120 respondents among smallholder dairy farmers in six wards purposively selected due to having larger number of dairy cattle was conducted. Likewise, 20 samples of animal feeds and 25 composite milk samples from among 120 interviewed farmers were analysed for aflatoxin B1 and M1 using High-Performance Liquid Chromatography (HPLC).&#x0D; Results: The results indicates that, aflatoxin B1 in feeds was 55%, with mean concentration of 8.6 ±5.9 μg/kg ranging from the lowest limit of detection (LOD) to 21.1 μg/kg with 5% samples above the maximum level stipulated by the Tanzania Bureau of Standards (TBS) and European Union (EU) of 20 μg/kg. Likewise, about 44% of the milk samples revealed to contain traces of aflatoxin M1 with a mean amount of 0.07 ±0.08 μg/L, ranging from the limit of detection (&lt;LOD) to 0.23 μg/L, and 12% exceeding the TBS and EU standards of 0.05 μg/kg.&#x0D; Conclusion: There is need to scale up aflatoxin awareness campaigns and regular monitoring throughout the food system in Tanzania.&#x0D; GRAPHICAL ABSTRACT: &#x0D;

222 High Throughput Screening of Bovine Mastitis from Raw Milk Using Maldi-Tof Ms and Machine Learning

Abstract To prevent pathogenic microorganisms associated with mastitis from entering the food supply, raw milk must be screened. This study investigated whether MALDI-TOF-MS combined with machine learning algorithms is a cost-effective method for screening dairy milk samples at high throughput. Raw milk from animals was analyzed using matrix-assisted laser desorption ionization mass spectrometry. Mass spectra were obtained for a training dataset of composite milk samples (n = 226) collected from a local dairy. The peaks from the mass spectra were then transported into a machine learning model, which detected non-obvious patterns that correspond with the mastitis condition. Finally, the diagnostic accuracy of the new model was evaluated using a separate set of milk samples (scoring set; n = 100). For optimal performance, decision trees, random forest models, gradient boosted trees, and neural network models were investigated. The results show that the gradient boosted decision tree model performs best for mastitis diagnosis, with simultaneous sensitivity and specificity greater than 0.8. The decision tree approach is conceptually straightforward and identifies candidate biomarker molecules indicative of mastitis. The combined MALDI TOF plus machine learning approach is effective for detecting mastitis in dairy cows and is likely a viable option for diagnosing a wide range of animal health issues. The MALDI TOF method is suitable for high-throughput screening because it only requires a microliter of inexpensive reagents.

Associations between early lactation intramammary infections and udder health and performance during the first 180 days in milk in first-lactation organic dairy cows

Prior data from our group showed that first-lactation cows under organic management in United States have a high prevalence of Staphylococcus aureus, Streptococcus spp., and Staphylococcus chromogenes intramammary infections (IMI) in early lactation. Nonetheless, the relationship between IMI, udder health, and milk production in organically reared primiparous cows remains elusive. The objectives of this observational study were to investigate the relationship between presence and persistence of IMI in the first 35 d in milk (DIM) and somatic cell count (SCC) and milk production during the first 6 mo of lactation on first-lactation organic dairy cows. The analysis included a total of 1,348 composite milk samples collected during the first 35 DIM that were submitted for milk culture and 1,674 Dairy Herd Improvement Association (DHIA) tests during the first 180 DIM from 333 heifers in 4 organic dairy farms, enrolled between February 2019 and January 2020. The association between IMI in the first 35 DIM and new high SCC (SCC > 200,000 cells/mL) and milk production during the first 6 mo of lactation was investigated using Cox proportional hazards regression and mixed linear regression, respectively. The association between IMI persistence (harboring the same microorganism as reported by the laboratory for 2 or more samples) in the first 35 DIM and number of DHIA tests with high SCC during the first 6 mo of lactation was modeled using negative binomial regression. The presence of IMI by Staph. aureus (hazard ratio [HR] [95% confidence interval {CI}]: 3.35 [2.64, 4.25]) or Streptococcus spp. (HR [95% CI]: 2.25 [2.12, 2.39]) during the first 35 DIM was associated with an increased risk of new high SCC during the first 6 mo of lactation. Milk production was reduced when Streptococcus spp. were identified in milk samples. However, there was no evidence of a difference in milk production in Staph. aureus IMI. Isolation of non-aureus staphylococci and mammaliicocci was related to a mild increase in the hazards of high SCC (HR [95% CI]: 1.34 [0.97, 1.85]) and a decrease in milk production during one or more postpartum tests. Presence of gram-negative or Streptococcus-like organisms IMI was not associated with either high SCC or milk production. Presence of Bacillus IMI was associated with a lower hazard of new high SCC (HR [95% CI]: 0.45 [0.30, 0.68]), and higher milk production during the first 180 d of lactation (overall estimate [95% CI]: 1.7 kg/d [0.3, 3.0]). The persistence of IMI in the first 35 DIM was associated with the number of tests with high SCC during the lactation for all microorganisms except for Staphylococcus chromogenes. Therefore, our results suggest that the persistence of IMI in the first 35 DIM could be an important factor to understand the association between IMI detected in early lactation and lactational SCC and milk production in organic dairy heifers. Our study described associations between IMI, udder health, and milk production in first-lactation organic dairy cows that are consistent with findings from conventional dairy farms.

Acute phase reaction to lipopolysaccharide-induced mastitis in early lactation dairy cows fed nitrogenic, glucogenic, or lipogenic diets

The availability of certain macronutrients is likely to influence the capacity of the immune system. Therefore, we investigated the acute phase response to intramammary (i.mam.) lipopolysaccharide (LPS) in dairy cows fed a nitrogenic diet (n = 10) high in crude protein, a glucogenic diet (n = 11) high in carbohydrates and glucogenic precursors, or a lipogenic diet (n = 11) high in lipids. Thirty-two dairy cows were fed one of the dietary concentrates directly after calving until the end of trial at 27 ± 3 days in milk (mean ± standard deviation). In wk 3 of lactation, 20 µg of LPS was i.mam. injected in one quarter, and sterile NaCl (0.9%) in the contralateral quarter. Milk samples of the LPS-challenged and control quarter were taken hourly from before (0 h) until 9 h after LPS challenge and analyzed for milk amyloid A (MAA), haptoglobin (HP), and IL-8. In addition, blood samples were taken in the morning, and composite milk samples at morning and evening milkings, from 1 d before until 3 d after LPS challenge, and again on d 9, to determine serum amyloid A (SAA) and HP in blood, and MAA and HP in milk. The mRNA abundance of various immunological and metabolic factors in blood leukocytes was quantified by quantitative reverse-transcription PCR from samples taken at -18, -1, 6, 9, and 23 h relative to LPS application. The dietary concentrates did not affect any of the parameters in blood, milk, and leukocytes. The IL-8 was increased from 2 h, HP from 2 to 3 h, and MAA from 6 h relative to the LPS administration in the milk of the challenged quarter and remained elevated until 9 h. The MAA and HP were also increased at 9 h after LPS challenge in whole-udder composite milk, whereas HP and SAA in blood were increased only after 23 h. All 4 parameters were decreased again on d 9. Similar for all groups, the mRNA abundance of HP and the heat shock protein family A increased after the LPS challenge, whereas the mRNA expression of the tumor necrosis factor α and the leukocyte integrin β 2 subunit (CD18) were decreased at 6 h after LPS challenge. The glucose transporter (GLUT)1 mRNA abundance decreased after LPS, whereas that of the GLUT3 increased, and that of the GLUT4 was not detectable. The mRNA abundance of GAPDH was increased at 9 h after LPS and remained elevated. The acute phase protein response was detected earlier in milk compared with blood indicating mammary production. However, immunological responses to LPS were not affected by the availability of specific macronutrients provided by the different diets.

Comparison of non-aureus staphylococcal and mammaliicoccal species found in both composite milk and bulk-tank milk samples of dairy cows collected in tandem

Non-aureus staphylococci and the closely related mammaliicoccal species (NASM) are the most common causes of bovine subclinical mastitis on modern dairy farms and are highly prevalent in bulk-tank milk. The purpose of this study was to determine the distribution of NASM in both composite cow milk (CCM) and bulk-tank milk (BTM) samples collected in tandem in commercial Flemish dairy herds and to estimate the origin of the different (subgroups of) NASM species present in BTM by applying strain typing (random amplification of polymorphic DNA or random amplified DNA [RAPD]). A single cross-sectional sampling was performed over 5 herds that volunteered to participate in the study. Composite cow milk samples (n = 356) were collected from all lactating cows (except those with clinical mastitis) during a milking in tandem with 6 BTM samples per herd sequentially collected immediately post that milking (n = 30). In total, 421 and 80 NASM isolates were recovered and identified by MALDI-TOF mass spectrometry from the CCM and BTM samples, respectively and a total of 21 and 12 different NASM species were identified from CCM and BTM samples, respectively. Staphylococcus cohnii was the most prevalent NASM species found in BTM followed by Staphylococcus haemolyticus, Staphylococcus epidermidis, Mammaliicoccus lentus, and Staphylococcus equorum, whereas from CCM samples the most common species were S. hemolyticus, S. cohnii, S. equorum, S. epidermidis, and Staphylococcus chromogenes. The prevalent NASM species in both CCM and BTM samples was distinct for each herd, corroborating other studies observing a herd-specific NASM microbiota. Random amplified DNA analysis was performed on 9 NASM species (S. chromogenes, S. epidermidis, S. haemolyticus, S. equorum, Mammaliicoccus sciuri, Staphylococcus xylosus, S. cohnii, Staphylococcus debuckii, and M. lentus) because these species were isolated from both sample types in a herd. The same RAPD types were found in both sample types for all NASM species selected for strain typing in varying degrees. When assessing the distribution of NASM species, differences within NASM species should be examined meaning a closer look should be taken at the strain level rather than at the species level only.

Mastitis on selected farms in Wakiso district, Uganda: Burden, pathogens and predictors of infectivity of antimicrobial resistant bacteria in dairy herds.

Mastitis and associated antimicrobial resistance (AMR) are major challenges to the dairy industry worldwide. This study aimed to expose the mastitis burden, causative bacteria and drivers for mastitis-causing multi-drug-resistant (MDR) Staphylococci infectivity in cows on dairy farms in Wakiso district, Uganda. On 22 farms, practices were documented using questionnaires, and 175 cows were screened by the California mastitis test. Composite milk samples from the positive reactors were submitted to the laboratory for bacterial culture testing. Antimicrobial sensitivity testing by the Kirby Bauer disc diffusion method was done only on Staphylococci with a panel of 10 antimicrobials of clinical relevance. Mastitis was detected in 80.6% (n=141) of the 175 sampled cows, of which sub-clinical mastitis (76.0%: n=133) was predominant. The Chi-squared analysis hypothesized that cow age (p=0.017), sub-county (p=0.013), parity (p<0.0001), sex of farm owner (p=0.003), farm duration in dairy production (p=0.048) and the use of milking salve (p=0.006) were associated with mastitis. Coagulase-negative Staphylococci were the most prevalent (71.4%; n=95), followed by Staphylococcus aureus (30.1%, n=40). Staphylococci (76.3%; n=135) were majorly resistant to penicillin and tetracycline. Only one isolate was phenotyped as a methicillin-resistant Staphylococcus specie (MRSS). The prevalences of MDR strains at cow and isolate level were 6.3% and 8.3%. The major MDR phenotype identified was penicillin-tetracycline-trimethoprim-sulphamethoxazole. The isolate detected as an MRSS exhibited the broadest MDR pattern. Cow parity was identified as a predictor of infectivity of mastitis-causing MDR Staphylococci in dairy herds. The high prevalence of mastitis and associated pathogen AMR found exposes possibilities of economic losses for the dairy sector warranting the need for farmer sensitization on the institution of proper mastitis prevention and control programs, with emphasis on milking hygiene practices and routine disease monitoring.

A randomized controlled trial to study the effects of an automated premilking stimulation system on milking performance, teat tissue condition, and udder health in Holstein dairy cows

The objectives were to examine the effect of an automated premilking stimulation (APS) by means of a high pulsation frequency (300 cycles/min) without a reduction of the vacuum in the pulsation chamber or claw piece on (1) milking performance, (2) teat tissue condition, and (3) udder health in dairy cows. In a randomized controlled field study, Holstein cows (n = 427) from 1 commercial dairy farm with a milking schedule of 3 times per day were assigned to treatment and control groups over a 90-d period. Treatments consisted of a maximum of 80 s (APS80) or 99 s (APS99) of mechanical stimulation at a pulsation rate of 300 pulses per minute and a ratio of 25:75 (no reduction of the pulsation chamber or milking vacuum). Cows in the control group (CON) received traditional premilking stimulation by means of manual forestripping for 8 s. Milking characteristics were documented with on-farm milk meters. Short- and long-term changes in teat tissue condition induced by machine milking were assessed visually on a weekly basis. Composite milk samples were analyzed once per month to determine somatic cell count. Generalized linear mixed models were used to study the effect of the treatment on the outcome variables. We observed no meaningful differences in milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the APS80, APS99, and CON groups were 13.5 (13.1-14.0), 13.2 (12.8-13.7), and 13.2 (12.8-13.7) kg for milk yield and 222 (213-231), 219 (210-228), and 223 (214-232) s for milking unit-on time, respectively. The effect of treatment on bimodality was modified by milk yield such that the odds of bimodality increased in the treatment groups with increasing milk yield. Compared with cows in the CON group, the odds ratios (95% CI) of bimodality were 1.08 (0.62-1.89) in the APS80 group and 0.89 (0.55-1.42) in the APS99 group at a milk yield of 11 kg and 2.0 (1.24-3.22) in the APS80 group and 2.08 (1.29-3.35) in the APS99 group at a milk yield of 16 kg. We observed differences in short- and long-term changes in teat tissue condition between the treatment and control groups. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.87 (1.35-2.58) for the APS80 group and 1.49 (1.08-2.07) for the APS99 group, and the odds of long-term changes were 1.52 (1.24-1.85) for cows in the APS80 group and 1.59 (1.31-1.94) for cows in the APS99 group. The least squares means (95% CI) for somatic cell counts (log10-transformed) were 4.74 (4.68-4.81) for the APS80 group, 4.77 (4.71-4.83) for the APS99 group, and 4.79 (4.73-4.86) for the CON group. We conclude that the APS system tested here had no negative effects on milk yield or milking unit-on time. However, differences in bimodality and teat tissue condition suggest that the APS system did not provide sufficient stimulation to facilitate a gentle milk harvest and adversely affects teat tissue condition.

Biochemical and Computational Assessment of Acute Phase Proteins in Dairy Cows Affected with Subclinical Mastitis.

Subclinical mastitis (SCM) is a predominant form of mastitis wherein major visible signs of disease are absent. The present study aimed to determine acute phase proteins (APPs) like ferritin, C-reactive protein (CRP), and microalbumin (Malb) in 135 composite milk and serum samples of healthy (n = 25) and SCM (n = 110) cows. As bovine mastitis is an inflammatory disease, the present study also aimed at finding novel anti-inflammatory compounds from natural sources by repurposing approach using computational studies. The findings of the present study revealed substantial elevation (p < 0.001) in milk SCC and an increase in ferritin, CRP, and Malb (p < 0.001) in milk and sera of the SCM group as compared to healthy animals. Receiver operating characteristics of milk SCC, milk, and serum APPs unraveled statistically substantial alteration (p < 0.001). Further, SCC was correlated with milk APPs ferritin (r = 0.26 **, p < 0.002), CRP (r = 0.19 *, p < 0.02), and Malb (r = 0.21 *, p < 0.01). Additionally, milk SCC was correlated with serum ferritin (r = 0.28 **, p < 0.001), CRP (r = 0.16, p > 0.05), and Malb (r = 0.16, p > 0.05). The findings of molecular docking revealed that Chaetoglobosin U was the most effective molecule that showed the highest binding affinity (kcal/mol) of -10.1 and -8.5 against ferritin and albumin. The present study concluded that the estimation of cow-side tests, SCC, and APPs in milk/serum is suitable to detect SCM and screening herd community. Furthermore, Chaetoglobosin U could be developed as a promising anti-inflammatory inhibitor; however, further studies are required to validate these findings.

Effect of dietary seaweed (Ascophyllum nodosum) supplementation on milk mineral concentrations, transfer efficiency, and hematological parameters in lactating Holstein cows

This study investigated the effect of feeding seaweed (Ascophyllum nodosum) to dairy cows on milk mineral concentrations, feed-to-milk mineral transfer efficiencies, and hematological parameters. Lactating Holstein cows (n = 46) were allocated to 1 of 2 diets (n = 23 each): (1) control (CON; without seaweed) and (2) seaweed (SWD; replacing 330 g/d of dried corn meal in CON with 330 g/d dried A. nodosum). All cows were fed the CON diet for 4 wk before the experiment (adaptation period), and animals were then fed the experimental diets for 9 wk. Samples included sequential 3-wk composite feed samples, a composite milk sample on the last day of each week, and a blood sample at the end of the study. Data were statistically analyzed using a linear mixed effects model with diet, week, and their interaction as fixed factors; cow (nested within diet) as a random factor; and data collected on the last day of the adaptation period as covariates. Feeding SWD increased milk concentrations of Mg (+6.6 mg/kg), P (+56 mg/kg), and I (+1,720 μg/kg). It also reduced transfer efficiency of Ca, Mg, P, K, Mn, and Zn, and increased transfer efficiency of Mo. Feeding SWD marginally reduced milk protein concentrations, whereas there was no effect of SWD feeding on cows' hematological parameters. Feeding A. nodosum increased milk I concentrations, which can be beneficial when feed I concentration is limited or in demographics or populations with increased risk of I deficiency (e.g., female adolescents, pregnant women, nursing mothers). However, care should also be taken when feeding SWD to dairy cows because, in the present study, milk I concentrations were particularly high and could result in I intakes that pose a health risk for children consuming milk.

Staphylococcus microti Strains Isolated from an Italian Mediterranean Buffalo Herd.

S. microti is a new species among non-aureus staphylococci (NAS) frequently found in bovine milk samples and associated with subclinical mastitis (SCM). The aim of this study was to analyze the presence of S. microti in 200 composite milk samples and 104 milking parlor surface swabs collected at a buffalo farm in Southern Italy to define its presence in milk and a milking parlor environment. The samples were inoculated onto different agar plates, and the isolates were identified by MALDI-TOF MS. The strains identified as S. microti (54/304 samples, 17.8%) were collected, and their purified genomic DNA was subjected to PCR amplification and whole 16S rRNA gene sequencing. Furthermore, their phenotypic resistance profiles were evaluated by a disk diffusion method, and the genotypic characterization of the tetracycline resistance was performed for the tetM and tetK genes by multiplex PCR. Four and forty-seven S. microti isolates from milk samples of lactating animals with subclinical mastitis (SCM) and intramammary infection (IMI), respectively, and three isolates from milking parlor surfaces were recovered. The genomic DNA was purified from the bacterial isolates, and the amplification and sequencing of the 16S gene further supported the proteomic identification as S. microti. No clinical mastitis was detected in the herd during the study period. The antimicrobial susceptibility testing revealed a worrisome 100% resistance to tetracyclines, genotypically mediated by the tetM gene for all strains. This study highlights that S. microti may be commonly isolated from dairy buffalo milk and milking parlor equipment. Its association with SCM or IMI remains to be established.

The prevalence, antimicrobial sensitivity, and factors associated with camel mastitis in Isiolo County, Kenya.

The one-humped camels (Camelus dromedarius) adapt very well to arid and semi-arid (ASALs) environments and continue to thrive and produce milk even during severe droughts when cattle, sheep, and goats experience high mortalities. These attributes make the dromedaries very vital for the survival of pastoralists, especially in the ASALs of Africa. Mastitis is among the most important diseases of camels and can cause significant economic losses, thereby endangering the livelihoods of pastoralists. This study aimed to estimate the prevalence, risk factors, and antimicrobial sensitivity of mastitis-causing pathogens in lactating camels in Isiolo County, Kenya. This was a cross-sectional study conducted in July and August 2021. A questionnaire was administered to the camel keepers to capture data on herd-level factors. Lactating camels were then examined for any visible signs of clinical mastitis and as well as to capture data on other animal-level factors such as age, weight, and body condition score. A composite milk sample was collected aseptically from randomly selected camels in a herd. The samples were initially tested for somatic cell counts (SCC) using Somatos mini® automatic cell counter. Culture and sensitivity testing was then carried out on any milk sample that had SCC ≥ 200,000 cells/mL. The descriptive analysis was initially used to summarize the continuous and categorical farm and camel factors, while mixed regression models were used to explore the association between/across mastitis and the farm as well as camel-level factors. A total of 210 lactating camels from 23 herds were selected and sampled in this study. The average age of camel keepers was 48.3 ± 16.3 years and they were involved in rearing camels for a mean of about 14.3 ± 8.6 years. The total number of camels in a herd ranged from 10 to 287, with the mean total herd milk production being 34.5 ± 24.9 L/day. The mean daily milk production per camel was 2.8 ± 1.7 L while the range for days in milk was between 21 and 787 days. The average age of camel first calving and the inter-calving interval was 56.3 ± 9.9 and 27.7 ± 9.9 months, respectively. The median parity of the dam was three and the body condition score was two. About 39.7% (83/210) of the sampled camels had clinical mastitis during the current lactation. The overall prevalence of mastitis (SCC ≥ 200,000 cells/mL) in camels was 17.6%. The main pathogens isolated were Streptococcus and Staphylococcus bacteria. Streptococcus isolates were mainly sensitive to ampicillin and resistant to cefaclor. All 18 Staphylococcus isolates were sensitive to tetracycline, while 12/18 isolates were resistant to ampicillin. The factors that were significantly associated with mastitis were the age of the respondent (p = 0.038), the number of years involved in camel rearing (p = 0.024), presence of clinical mastitis since calving (p = 0.039), body condition score (p = 0.040), and age of the dam at the time of examination (p = 0.072). Results from this study revealed that mastitis is an important condition among camels in the pastoral communities of Isiolo County, Kenya. Thus, pastoralists should continue to be aware of and trained on mastitis occurrence and its control in the pastoral ecosystem to reduce potential economic losses.

Antimicrobial Resistance, Virulence Profiles, and Public Health Significance of Enterococcus faecalis Isolated from Clinical Mastitis of Cattle in Bangladesh.

This study was designed to identify Enterococcus faecalis from clinical mastitis of cattle and determine their antimicrobial resistance and virulence determinants to evaluate their potential public health significance. A total of 105 composite milk samples (80 from cattle with clinical mastitis and 25 from apparently healthy cattle) were analyzed. E. faecalis were isolated by culturing on enterococcal selective media and identified by PCR and sequencing. Antimicrobial resistance phenotype was elucidated by the disc diffusion method, and MIC was determined by broth microdilution method according to CLSI guidelines. Detection of antimicrobial resistance and virulence genes was done by PCR. E. faecalis were isolated from 11.25% (9/80) of the clinical mastitis and 4% (1/25) of the apparently healthy cattle milk samples. The disc diffusion test revealed 40% isolates as resistant to tetracycline and azithromycin, respectively. Among them, 20% (2/10) of isolates showed resistance to both tetracycline and azithromycin. Tetracycline-resistant isolates showed MIC ranging from ≥64 to >128 μg/ml and carried tetracycline-resistant genes tetK, tetL, and tetM in 25%, 25%, and 50% of the resistant isolates, respectively. On the other hand, all the isolates were sensitive to amoxicillin, ampicillin, bacitracin, chloramphenicol, gentamicin, penicillin, and vancomycin. In addition, the isolates carried at least one of the nine virulence genes screened with pil having the highest frequency, followed by fsrB, fsrC, ace, sprE, gelE, and agg genes. Positive correlations were evident between ace, fsrC, gelE, and sprE genes that are associated with the attachment and biofilm formation in E. faecalis. E. faecalis isolated in this study carried antibiotic resistance and virulence determinants which explain their competence to be potential human pathogens.

Comparison of cow milk components between daily actual and AM-PM composite samples from 2 consecutive milkings by Dairy Herd Improvement

This study was undertaken to evaluate the level of agreement of milk fat, protein, lactose, MUN, and SCC concentrations between daily actual and AM-PM composite milk samples taken at 2 consecutive DHI milkings and to assess factors affecting their level of agreement. Milk samples from 2 consecutive milkings were collected using in-line milk meters on 4,340 Holstein cows in 100 Canadian commercial dairy herds. Three milk samples per cow were analyzed for major components: (1) evening samples; (2) morning samples; and (3) an AM-PM composite sample obtained by visually mixing equal volumes of milk from 2 consecutive milkings. Daily actual milk component concentrations were computed proportionally to milk yields. Equal 50:50 composite milk component concentrations were the average of evening and morning samples. Concordance correlation coefficients (CCC) between actual and equal 50:50 samples varied from 0.997 to 1.000. For the comparison between daily actual and AM-PM composite milk component concentrations, CCC ranged from 0.911 to 0.964. This suggested that AM-PM composite samples were not always composed of an equal 50:50 volume of milk from evening and morning milkings. A great variation of CCC was observed between herds, indicating differences in their assessment of volumes of milk to pour into vials at each milkings. Assuming that milk samples were homogeneous, AM-PM composite samples predicted daily actual milk components with great precision and accuracy. However, this was not the case in all herds. Visually assessing milk volumes to pour into vials when creating AM-PM composite milk samples was the major cause of a decrease in level of agreement when predicting daily actual milk component concentrations, which varied between herds. One recommendation might be to add indicators on DHI vials to guide in mixing an equal milk volume from 2 consecutive milkings. Because DHI records are used in decision making, it is important that predicted daily milk component concentrations are as close as possible to daily actual milk component concentrations. Producers can make an informed decision on which sampling scheme to chose according to their management objectives.